Tissue engineering alms at the development of biological substitutes that restore, maintain or improve tissue function (Langer & Vacanti J. P. Tissue Engineering. Science. 260:920-926, 1993). One strategy currently adopted to regenerate new tissues such as skin, cartilage or bone, is the isolation, in vitro expansion and loading into a three-dimensional scaffold the expanded cells (Brittberg M. et al., Treatment of deep articular cartilage defects in the knee with autologous chondrocyte transplantation. N Engl J Med 331:889-895.1994; Quarto R. et al., Repair of large bone defects with the use of autologous bone marrow stromal cells. N Engl J Med 344:385-386, 2001; Rheinwald J. G. et al., Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell 6:331-344, 1975).
Bone marrow stromal cells (BMSC) have been used for the regeneration of bone and have been demonstrated to be expandable in monolayers from a marrow aspirate and, when loaded into a porous ceramic scaffold after expansion, are capable of generating an osteoinductive construct which supports bridging of large segmental defects in human (Quarto R. et al., Repair of large bone defects with the use of autologous bone marrow stromal cells. N Engl J Med 344:385-386, 2001).
A bioreactor has been developed allowing for the perfusion of cell suspensions through three-dimensional porous scaffolds (Wendt D. et al., Oscillating perfusion of cell suspensions through three-dimensional scaffolds enhances cell seeding efficiency and uniformity. Biotechnol Bioeng, 84:205-214, 2003). Cells were isolated from a cartilage biopsy or bone marrow aspirate and expanded in monolayers prior to use in the bioreactor. Controlled perfusion of BMSC, initially expanded in monolayers, has been demonstrated to increase the capacity of cells to differentiate and deposit mineralized matrix (Bancroft G. N. et al., Fluid flow increases mineralized matrix deposition in 3D perfusion culture of marrow stromal osteoblasts in a dose-dependent manner. Proc Natl Acad Sci USA 99:12600-12605, 2002). It has also been demonstrated that BMSC can be expanded in suspension cultures in spinner flasks, however, the disclosed method did not involve association of the expanded cells to a three-dimensional scaffold (Baksh D. et al., Adult human bone marrow derived mesenchymal progenitor cells are capable of adhesion independent survival and expansion. Exp Hematol 31:723-732, 2003).
GB Patent Application 2 178 447 discloses a cultivation system for cell attachment using a matrix material in which cells are proliferated along fibers of a sheet in three dimensions. The matrix material is provided in a configuration as a reactor through which separate conduits are placed therethrough, one conduit for the supply and/or removal of liquid medium, and the other for providing a supply of gases.
U.S. Pat. No. 6,372,495 discloses a reactor in which cells are seeded and allowed to distribute throughout the reactor and adhere to a solid support. The reactor is rotated along its longitudinal axis to distribute cells throughout the reactor to prevent the formation of a cell pellet. The cells are thus allowed to follow a circular path through the reactor so that the cells repeatedly come into contact with the matrix material and get entrapped therein.
While the aforementioned disclose various methods for seeding cells to a three-dimensional scaffold, it is desirable to develop a method and system that increases the efficiency and consistency of cell loading throughout a three-dimensional scaffold such that the cells can further expand in a more even manner throughout the scaffold, thus producing a more desirable tissue implant.